1. Field of the Invention
The present invention provides adeno-associated virus 5 (AAV5) and vectors derived therefrom. Thus, the present invention relates to AAV5 vectors for and methods of delivering nucleic acids to cells of subjects.
2. Background Art
Adeno associated virus (AAV) is a small nonpathogenic virus of the parvoviridae family (for review see 28). AAV is distinct from the other members of this family by its dependence upon a helper virus for replication. In the absence of a helper virus, AAV has been shown to integrate in a locus specific manner into the q arm of chromosome 19 (21). The approximately 5 kb genome of AAV consists of one segment of single stranded DNA of either plus or minus polarity. Physically, the parvovirus virion is non-enveloped and its icosohedral capsid is approximately 20-25 nm in diameter.
To date 8 serologically distinct AAVs have been identified and 6 have been isolated from humans or primates and are referred to as AAV types 1-6 (1). The most extensively studied of these isolates is AAV type 2 (AAV2). The genome of AAV2 is 4680 nucleotides in length and contains two open reading frames (ORFs), the right ORF and the left ORF. The left ORF encodes the non-structural Rep proteins, Rep40, Rep52, Rep68 and Rep78, which are involved in regulation of replication and transcription in addition to the production of single-stranded progeny genomes (5-8, 11, 12, 15, 17, 19, 21-23, 25, 34, 37-40). Furthermore, two of the Rep proteins have been associated with the preferential integration of AAV genomes into a region of the q arm of human chromosome 19. Rep68/78 have also been shown to possess NTP binding activity as well as DNA and RNA helicase activities. The Rep proteins possess a nuclear localization signal as well as several potential phosphorylation sites. Mutation of one of these kinase sites resulted in a loss of replication activity.
The ends of the genome are short inverted terminal repeats which have the potential to fold into T-shaped hairpin structures that serve as the origin of viral DNA replication. Within the ITR region two elements have been described which are central to the function of the ITR, a GAGC repeat motif and the terminal resolution site (TRS). The repeat motif has been shown to bind Rep when the ITR is in either a linear or hairpin conformation (7, 8, 26).
This binding serves to position Rep68/78 for cleavage at the TRS which occurs in a site- and strand-specific manner. In addition to their role in replication, these two elements appear to be central to viral integration. Contained within the chromosome 19 integration locus is a Rep binding site with an adjacent TRS. These elements have been shown to be functional and necessary for locus specific integration.
The AAV2 virion is a non-enveloped, icosohedral particle approximately 20-25 nm in diameter. The capsid is composed of three related proteins referred to as VP1, 2 and 3 which are encoded by the right ORF. These proteins are found in a ratio of 1:1:10 respectively. The capsid proteins differ from each other by the use of alternative splicing and an unusual start codon. Deletion analysis of has shown that removal or alteration of AAV2 VP 1 which is translated from an alternatively spliced message results in a reduced yield of infections particles (15, 16, 38). Mutations within the VP3 coding region result in the failure to produce any single-stranded progeny DNA or infectious particles (15, 16, 38).
The following features of the characterized AAVs have made them attractive vectors for gene transfer (16). AAV vectors have been shown in vitro to stably integrate into the cellular genome; possess a broad host range; transduce both dividing and non dividing cells in vitro and in vivo (13, 20, 30, 32) and maintain high levels of expression of the transduced genes (41). Viral particles are heat stable, resistant to solvents, detergents, changes in pH, temperature, and can be concentrated on CsCl gradients (1, 2). Integration of AAV provirus is not associated with any long term negative effects on cell growth or differentiation (3, 42). The ITRs have been shown to be the only cis elements required for replication, packaging and integration (35) and may contain some promoter activities (14).
AAV2 was originally thought to infect primate and non-primate cell types provided the appropriate helper virus was present. However, the inability of AAV2 to infect certain cell types is now known to be due to the particular cellular tropism exhibited by the AAV2 virus. Recent work has shown that some cell lines are transduced very poorly by AAV2 (30). Binding studies have indicated that heparin sulfate proteoglycans are necessary for high efficiency transduction with AAV2. AAV5 is a unique member of the parvovirus family. The present DNA hybridization data indicate a low level of homology with the published AAV1-4 sequences (31). The present invention shows that, unlike AAV2, AAV5 transduction is not effected by heparin as AAV2 is and therefore will not be restricted to the same cell types as AAV2.
The present invention provides a vector comprising the AAV5 virus or a vector comprising subparts of the virus, as well as AAV5 viral particles. While AAV5 is similar to AAV2, the two viruses are found herein to be physically and genetically distinct. These differences endow AAV5 with some unique properties and advantages which better suit it as a vector for gene therapy. For example, one of the limiting features of using AAV2 as a vector for gene therapy is production of large amounts of virus. Using standard production techniques, AAV5 is produced at a 10-50 fold higher level compared to AAV2. Because of its unique TRS site and rep proteins, AAV5 should also have a distinct integration locus compared to AAV2.
Furthermore, as shown herein, AAV5 capsid protein, again surprisingly, is distinct from AAV2 capsid protein and exhibits different tissue tropism, thus making AAV5 capsid-containing particles suitable for transducing cell types for which AAV2 is unsuited or less well-suited. AAV2 and AAV5 have been shown to be serologically distinct and thus, in a gene therapy application, AAV5, and AAV5-derived vectors, would allow for transduction of a patient who already possess neutralizing antibodies to AAV2 either as a result of natural immunological defense or from prior exposure to AAV2 vectors. Another advantage of AAV5 is that AAV5 cannot be rescued by other serotypes. Only AAV5 can rescue the integrated AAV5 genome and effect replication, thus avoiding unintended replication of AAV5 caused by other AAV serotypes. Thus, the present invention, by providing these new recombinant vectors and particles based on AAV5 provides a new and highly useful series of vectors.